Long Non-Specific PCR Products

Hello! I need help to optimize a PCR and will really appreciate your input.

This is a PCR with degenerate primers that we designed in our laboratory. The expected PCR product should be between 250-300 bp, depending of the specie.

In the first picture I use the following PCR conditions:

95ºC 3 min

95ºC 45 sec
40ºC 45 sec 35 cycles
72ºC 1 min

72ºC 5 min

The order in the gel

Marker
Sample Specie 1
Sample Specie 1
Positive Control (Specie that we know certainly that has to have 250 bp)
Negative Control

A looot of non specific bands appear in my samples. In the second picture I decreased the time for each of the steps, so it went like this:

95ºC 3 min

95ºC 30 sec
40ºC 30 sec 35 cycles
72ºC 45 sec

72ºC 5 min

No changes were done in the PCR mastermix reaction.
I want to know if there is any other change to my thermocycling conditions in order to get rid of the un specific bands but still mantain the expected band.

Your annealing temperature is way too low. I've never had a successful PCR when annealing below 48. If your primers have too low an annealing temperature, you must redesign your primers. The low annealing temperature will cause many spurious bands.

Besides redesigning the primers, if I would want to try smth in between the new primers coming, I would try with a annealing temperature 5 oC above the highest melting temperature of each primer; hopefully this is not below 48oC ...otherwise, you have no chance.

Thanks both of you for your comments. After reading your comments, and doing another try today, I do think that we need to redesign specific primers, as it has been impossible to get rid of the other bands. We will try to purifiy the band of interest and sequence it so we can design more specific primers.

And I will try the suggestion of rising the annealing temperature, the highest Tm is 49ºC for one primer and 52ºC for the other one, so it will be feasible to do it.
I will keep you posted about it.

Please try your PCR reaction with a 52 or 55 anneal step. Your primers may simply work. I wish I could just get people to ignore calculated annealing temperatures entirely. Instead they spend days calculating them instead of spending a few hours simply trying a few PCR test reactions. Most 20 bp primers work when you anneal at 55.